Green Bacterium Chloroflexus Aurantiacus Research Articles

ADAPTATION OF GREEN PHOTOSYNTHETIC BACTERIA TO DIFFERENT ILLUMINATION INTENSITY ACCORDING TO SPECTROSCOPY DATA ON CHLOROSOMES

The absorption and circular dichroism spectra of chlorosomes isolated from green photosynthetic bacteria Chloroflexus aurantiacus grown under di erent illumination were studied. It was found that as the intensity of the growth light decreases, the spectra shift to the red side and become narrower and larger in amplitude. Theoretical modeling of the data obtained was performed using the theory of excitons. The conclusion was formulated that the number of bacteriochlorophyll c molecules in linear chains, which form the basis of the elementary blocks of chlorosomes, become greater as the intensity of light at which bacteria are grown decreases. It was suggested that this phenomenon increases the e ciency of capturing weak light uxes and thereby increases the chances of bacterial survival in conditions of sunlight de ciency.

Carbonic Anhydrase in Anoxygenic Phototrophic Bacteria

The genes encoding carbonic anhydrase (CA) were found in all anoxygenic purple bacteria. The genes of α- and β-CA were found in purple nonsulfur bacteria of the class Alphaproteobacteria:Rhodospirillum rubrum, Rhodospirillum fulvum, Rhodoblastus acidophilus, and Rhodopseudomonas palustris. The alphaproteobacteria Rhodomicrobium vannielii, Blastochlorisviridis, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter veldkampii, Rhodovulumeuryhalinum, and Rhodovulum sulfidophilum possessed only the β-CA genes. Both nonsulfur purple bacteria of the class Betaproteobacteria (Rubrivivax gelatinosus) and purple sulfur bacteria (class Gammaproteobacteria) contained the α- and β-CA. No CA genes were found in green sulfur bacteria Chlorobaculum limnaeum and Chlorobaculum parvum, as well as in filamentous green nonsulfur bacteria Chloroflexus aurantiacus. However, the β-CA gene was revealed in Oscillochloris trichoides, which belonged to the latter taxonomic group. No γ-CA genes were detected in the genomes of the phototrophic bacteria studied in the present work. Although CA genes were present in all purple bacteria, the α- and β-CA activity was observed only in four species of purple nonsulfur Alphaproteobacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodoblastusacidophilus, and Rhodospirillum fulvum. These bacteria are able to synthesize CA under both photoautotrophic and photoheterotrophic conditions in the media with acetate, malate, or fructose. These bacteria (except for Rhodospirillum fulvum, which is unable to grow under aerobic conditions), also exhibit CA activity when grown under aerobic conditions in the dark.

Variability of aggregation extent of light-harvesting pigments in peripheral antenna of Chloroflexus aurantiacus.

The stationary ground state and femtosecond time-resolved absorption spectra as well as spectra of circular dichroism were measured at room temperature using freshly prepared samples of chlorosomes isolated from fresh cultures of the green bacterium Chloroflexus aurantiacus. Cultures were grown by using as inoculum the same seed culture but under different light conditions. All measured spectra clearly showed the red shift of BChl c Qy bands (up to 5nm) for low-light chlorosomes as compared to high-light ones, together with concomitant narrowing of these bands and increasing of their amplitudes. The sizes of the unit BChl c aggregates of the high-light-chlorosomes and the low-light ones were estimated. The fit of all experimental spectra was obtained within the framework of our model proposed before (Fetisova et al., Biophys J 71:995-101, 1996). The model assumes that a unit building block of the BChl c antenna has a form of a tubular aggregate of L = 6 linear single or double exciton-coupled pigment chains within a rod element, with the pigment packing density, approximating that in vivo. The simultaneous fit of all experimental spectra gave the number of pigments in each individual linear pigment chain N = 4 and N = 6 for the high-light and the low-light BChl c unit building blocks, respectively. The size of a unit building block in the BChl c antenna was found to vary from L × N = 24 to L × N = 36 exciton-coupled BChl c molecules being governed by the growth-light intensity. All sets of findings for Chloroflexus aurantiacus chlorosomes demonstrated the biologically expedient light-controlled variability, predicted by us, of the extent of BChl c aggregation within a unit building block in this antenna.

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus.

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52°C (an optimal growth temperature) and 44°C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44°C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44°C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.

Femtosecond charge separation in dry films of reaction centers of Rhodobacter sphaeroides and Chloroflexus aurantiacus

In this work, the influence of the crystallographic water on electron transfer between primary donor P and acceptor B(A) was studied in reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides and the green bacterium Chloroflexus aurantiacus. For this purpose, time constants and oscillations of charge separation kinetics are compared between dry film RCs and RCs in glycerol-water buffer at 90 K. A common result of the drying of Rba. sphaeroides and Cfx. aurantiacus RCs is slowing of the charge separation process, decrease in amplitude of the oscillatory components of the kinetics, and the depletion of its spectrum. Thus, the major time constant of stimulated emission decay of P* bacteriochlorophyll dimer at 940 nm is increased from 1.1 psec for water-containing Rba. sphaeroides RCs to 1.9 psec for dry films of Rba. sphaeroides RCs. An analogous increase from 3.5 to 4.2 psec takes place in Cfx. aurantiacus RCs. In dry films of Rba. sphaeroides RCs, the amplitude of coherent oscillations of the absorption band of monomeric bacteriochlorophyll B(A)(-) at 1020 nm is 1.8 times less for the 130-cm(-1) component and 2.3 times less for the 32-cm(-1) component than the analogous amplitudes for water-containing RCs. Measurements in the analogous band of Cfx. aurantiacus RCs show that strong decrease (~5-10 times) of the B(A)(-) absorption band and strong slowing (from ~0.8 to ~3 psec) of B(A)(-) accumulation together with ~3-fold decrease in oscillation amplitude occurs on drying of these RCs. The overtones of the 32-cm(-1) component disappeared from the oscillations of the kinetics at 940 and 1020-1028 nm after drying of the Rba. sphaeroides and Cfx. aurantiacus RCs. The results are in agreement with the results for GM203L mutant of Rba. sphaeroides, in which the HOH55 water molecule is sterically removed, and with the results for dry films of pheophytin-modified RCs of Rba. sphaeroides R-26 and for YM210W and YM210L Rba. sphaeroides mutant RCs. The data are discussed in terms of the influence (or participation) of the HOH55 water molecule on electron transfer along the chain of polar atomic groups N-Mg(P(B))-N-C-N(HisM202)-HOH55-O=(B(A)) connecting P(B) and B(A) in Rba. sphaeroides RCs.

Temperature and Ionic Strength Effects on the Chlorosome Light-Harvesting Antenna Complex

Chlorosomes, the peripheral light-harvesting antenna complex from green photosynthetic bacteria, are the largest and one of the most efficient light-harvesting antenna complexes found in nature. In contrast to other light-harvesting antennas, chlorosomes are constructed from more than 150,000 self-assembled bacteriochlorophylls (BChls) and contain relatively few proteins that play secondary roles. These unique properties have led to chlorosomes as an attractive candidate for developing biohybrid solar cell devices. In this article, we investigate the temperature and ionic strength effects on the viability of chlorosomes from the photosynthetic green bacterium Chloroflexus aurantiacus using small-angle neutron scattering and dynamic light scattering. Our studies indicate that chlorosomes remain intact up to 75 °C and that salt induces the formation of large aggregates of chlorosomes. No internal structural changes are observed for the aggregates. The salt-induced aggregation, which is a reversible process, is more efficient with divalent metal ions than with monovalent metal ions. Moreover, with treatment at 98 °C for 2 min, the bulk of the chlorosome pigments are undamaged, while the baseplate is destroyed. Chlorosomes without the baseplate remain rodlike in shape and are 30-40% smaller than with the baseplate attached. Further, chlorosomes are stable from pH 5.5 to 11.0. Together, this is the first time such a range of characterization tools have been used for chlorosomes, and this has enabled elucidation of properties that are not only important to understanding their functionality but also may be useful in biohybrid devices for effective light harvesting.

SANS Investigation of the Photosynthetic Machinery of Chloroflexus aurantiacus

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.

Excitation energy transfer in isolated chlorosomes from Chloroflexus aurantiacus

Chlorosomes from green photosynthetic bacteria Chloroflexus aurantiacus have been studied by time-resolved femtosecond transient absorption spectroscopy. The fastest kinetics of 200–300 fs resolved, was interpreted to stem for intra-chlorosomal excitation energy transfer. Energy transfer from the antenna to the baseplate appeared as a major 9.2 ps rise component detected at the baseplate probe wavelength. Excitation energy transfer rates were evaluated for a model chlorosome. Calculated rod to rod, and rods to baseplate rate constants of 200–400 fs and 10–20 ps, respectively, are in accord with the experimental results.

The search for an optimal orientational ordering of Q y transition dipoles of subantenna molecules in the superantenna of photosynthetic green bacteria: Model calculations

This work continues a series of our investigations on efficient strategies of functioning of natural light-harvesting antennae, initiated by our concept of rigorous optimization of photosynthetic apparatus structure by functional criterion. Using computer modeling for the functioning of the natural antennae, we suggested some basic principles for designing optimal model systems. Targeted searches for these principles in in vivo systems allowed us to recognize some of them in natural antennae. This work deals with the problem of the structure optimization of nonuniform superantennae of photosynthetic green bacteria. These superantennae consist of several uniform subantennae which produces a problem of their optimal coordination. In this work, we used mathematical modeling for the functioning of these natural superantennae to consider a possible way to optimize the superantenna structure using optimization of mutual spatial orientation of Qy transition dipoles of subantennae pigments. This allowed us to determine some modes of optimal orientational ordering of Qy transition dipoles of subantennae pigments in the model of the green bacterium Chloroflexus aurantiacus superantenna. It was shown that the optimal mutual orientation of Qy transition dipoles of subantennae pigments (resulting in stable minimizing of the energy transfer time within the superantenna and, as a consequence, in decrease in energy losses) ensures the high efficiency and stability of the superatenna functioning.

Microbial Synthesis of Poly((R)-3-hydroxybutyrate-co- 3-hydroxypropionate) from Unrelated Carbon Sources by Engineered Cupriavidus necator

Cupriavidus necator was engineered aiming to synthesize poly[(R)-3-hydroxybutyrate-co-3-hydroxypropionate] copolyester, P(3HB-co-3HP), from structurally unrelated carbon sources without addition of any precursor compounds. We modified a metabolic pathway in C. necator for generation of 3-hydroxypropionyl-CoA (3HP-CoA) by introducing malonyl-CoA reductase and the 3HP-CoA synthetase domain of trifunctional propionyl-CoA synthase; both members of the 3-hydroxypropionate cycle, a novel CO(2)-fixation pathway in the green nonsulfur bacterium Chloroflexus aurantiacus . In this recombinant strain, 3HP-CoA was expected to be provided from acetyl-CoA via malonyl-CoA, and then copolymerized by the function of polyhydroxyalkanoate synthase along with (R)-3-hydroxybutyryl-CoA synthesized from two acetyl-CoA molecules. C. necator wild-type strains H16 and JMP134 harboring the two heterologous genes actually synthesized P(3HB-co-3HP) copolyester with 0.2-2.1 mol % of 3HP fraction from fructose or alkanoic acids of even carbon numbers. Enzyme assay suggested that lower activity of 3HP-CoA synthetase than that of malonyl-CoA reductase caused the limited incorporation of 3HP unit into the copolyesters synthesized by the recombinant strains. The present study demonstrates the potential of engineering metabolic pathways for production of copolyesters having favorable characteristics from inexpensive carbon resources.

Purification, characterization and crystallization of menaquinol:fumarate oxidoreductase from the green filamentous photosynthetic bacterium Chloroflexus aurantiacus

The integral membrane protein complex, menaquinol:fumarate oxidoreductase (mQFR) has been purified, identified and characterized from the thermophilic green filamentous anoxygenic photosynthetic bacterium Chloroflexus aurantiacus. The complex is composed of three subunits: a 74 kDa flavoprotein that contains a covalently bound flavin adenine dinucleotide, a 28 kDa iron-sulfur cluster-containing polypeptide, and a 27 kDa transmembrane polypeptide, which is also the binding site of two b-type hemes and two menaquinones. The purified complex has an apparent molecular mass of 260 kDa by blue-native PAGE, which is indicative of a native homodimeric form. The isolated complex is active in vitro in both fumarate reduction and succinate oxidation. It has been analyzed by visible absorption, redox titration, chemical analysis and EPR spectroscopy. In addition, phylogenetic analysis shows that the QFR of both C. aurantiacus and Chlorobium tepidum are most closely related to those found in the delta-proteobacteria. The purified enzyme was crystallized and X-ray diffraction data obtained up to 3.2 Å resolution.

Immobilization of Functional Light Antenna Structures Derived from the Filamentous Green Bacterium Chloroflexus aurantiacus

The integration of highly efficient, natural photosynthetic light antenna structures into engineered systems while their biophotonic capabilities are maintained has been an elusive goal in the design of biohybrid photonic devices. In this study, we report a novel technique to covalently immobilize nanoscaled bacterial light antenna structures known as chlorosomes from Chloroflexus aurantiacus on both conductive and nonconductive glass while their energy transducing functionality was maintained. Chlorosomes without their reaction centers (RCs) were covalently immobilized on 3-aminoproyltriethoxysilane (APTES) treated surfaces using a glutaraldehyde linker. AFM techniques verified that the chlorosomes maintained their native ellipsoidal ultrastructure upon immobilization. Results from absorbance and fluorescence spectral analysis (where the Stokes shift to 808/810 nm was observed upon 470 nm blue light excitation) in conjunction with confocal microscopy confirm that the functional integrity of immobilized chlorosomes was also preserved. In addition, experiments with electrochemical impedance spectroscopy (EIS) suggested that the presence of chlorosomes in the electrical double layer of the electrode enhanced the electron transfer capacity of the electrochemical cell. Further, chronoamperometric studies suggested that the reduced form of the Bchl- c pigments found within the chlorosome modulate the conduction properties of the electrochemical cell, where the oxidized form of Bchl- c pigments impeded any current transduction at a bias of 0.4 V within the electrochemical cell. The results therefore demonstrate that the intact chlorosomes can be successfully immobilized while their biophotonic transduction capabilities are preserved through the immobilization process. These findings indicate that it is feasible to design biophotonic devices incorporating fully functional light antenna structures, which may offer significant performance enhancements to current silicon-based photonic devices for diverse technological applications ranging from CCD devices used in retinal implants to terrestrial and space fuel cell applications.

Optimal coupling of subantennas as a strategy for efficient functioning of the light-harvesting antennas in photosynthesizing organisms: Model computations

This work continues the cycle of studies into the strategy of efficient functioning of the natural light-harvesting molecular antennas within the framework of the previously proposed concept of stringent optimization of the structure of the photosynthesizing machinery according to the functional criterion [1]. The presence of inhomogeneous antennas composed of different subantennas poses the problem of their coupling [2]. One of the structural optimizing factors is the relative orientation of Q y transition dipoles of the light-harvesting pigments in subantennas [3]. This paper briefs the mathematical modeling of the dynamics of excitation energy transfer in the superantenna of the photosynthesizing green bacteria Chloroflexus ( Cf. ) aurantiacus. This allowed us to compute the optimal relative orientation of the Q y transition dipoles in subantenna molecules using the formalism of probability matrices and to demonstrate the significance of their optimal coupling for an efficient and stable functioning of the superantenna. The superantenna of Cf. aurantiacus is localized to three subcellular structures: chlorosomes, containing the oligomeric BChl c subantenna B740; the baseplate, containing the monomeric BChl a subantenna B798; and cytoplasmic membrane, containing the BChl a subantenna B808‐866, analogous to the subantenna B800‐ 850 of purple bacteria [4]. The excitation energy transfer B740 → B798 → B808 → B866 proceeds according to the thermodynamic potential [5]. The Q y transition dipoles in BChl c clusters of subantenna B740 are mutually parallel and oriented along the long axis of the chlorosome [6], whereas the Q y transition dipoles in B808 and B866 subantenna molecules make angles of ~43° and ~8° with the plane of the membrane, respectively [7]. The orientation of Q y transition dipoles for the intermediate subantenna B798 in BChl ‡ is unknown. In this work, we modeled the energy transfer B740 → B798 → B808 to analyze the optimization of this process by optimizing the orientation of Q y transition dipoles in BChl ‡ subantenna B798. An infinite three-dimensional antenna displaying

Femtosecond Spectroscopy of the Primary Charge Separation in Reaction Centers ofChloroflexusaurantiacuswith Selective Excitation in the QYand Soret Bands

The primary charge separation and electron-transfer processes of photosynthesis occur in the reaction center (RC). Isolated RCs of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus were studied at room temperature by using femtosecond transient absorption spectroscopy with selective excitation. Upon excitation in the Q(Y) absorbance band of the bacteriochlorophyll (BChl) dimer (P) at 865 nm, a 7.0 +/- 0.5 ps kinetic component was observed in the 538 nm region (Q(X) band of the bacteriopheophytin (BPheo)), 750 nm region (Q(Y) band of the BPheo), and 920 nm region (stimulated emission of the excited-state of P), indicating that this lifetime represents electron transfer from P to BPheo. The same time constant was also observed upon 740 nm or 800 nm excitation. A longer lifetime (300 +/- 30 ps), which was assigned to the time of reduction of the primary quinone, Q(A), was also observed. The transient absorption spectra and kinetics all indicate that only one electron-transfer branch is involved in primary charge separation under these excitation conditions. However, the transient absorption changes upon excitation in the Soret band at 390 nm reveal a more complex set of energy and electron-transfer processes. By comparison to studies on the RCs of the purple bacterium Rhodobacter sphaeroides, we discuss the possible mechanism of electron-transfer pathway dependence on excitation energy and propose a model of the Cf. aurantiacus RC that better explains the observed results.

Occurrence of Hydrogenases in Cyanobacteria and Anoxygenic Photosynthetic Bacteria: Implications for the Phylogenetic Origin of Cyanobacterial and Algal Hydrogenases

Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.

Transmission electron microscopic study on supramolecular nanostructures of bacteriochlorophyll self-aggregates in chlorosomes of green photosynthetic bacteria

Supramolecular nanostructures of bacteriochlorophyll (BChl) self-aggregates in major light-harvesting complexes (chlorosomes) of green photosynthetic bacteria were successfully observed by freeze-fracture transmission electron microscope. Rod-shaped nanostructures with approximately 10 nm in diameter could be visualized in three types of green sulfur bacteria (Chlorobium). Diameter of the rod-shaped nanostructures in Chlorobium chlorosomes was independent of the molecular structures of their light-harvesting pigments, namely BChl-c or d. In contrast, chlorosomes of the green filamentous bacterium Chloroflexus aurantiacus had rod-shaped nanostructures with approximately 5 nm in diameter. The present results support that BChl self-aggregates in chlorosomes form rod-shaped nanostructures called rod-elements with approximately 10- and 5-nm diameters for Chlorobium and Chloroflexus, respectively.

Some factors controlling the biosynthesis of chlorosome antenna bacteriochlorophylls in green filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae

We determined the concentrations of bacteriochlorophylls (BChl) in the light-harvesting antennae of Oscillochloris trichoides (of the family Oscillochloridaceae belonging to green filamentous mesophilic bacteria) cultivated either with gabaculine, an inhibitor of the C-5 pathway of BChl biosynthesis in a number of bacteria, or at various illumination intensities. We determined the BChl c: BChl a molar ratios in intact cells, in chlorosome-membrane complexes, and in isolated chlorosomes. We revealed that BChl c synthesis in Osc. trichoides was more gabaculine-sensitive than BChl a synthesis. Accordingly, an increase in gabaculine concentrations in the medium resulted in a decrease in the BChl c: BChl a ratio in the tested samples. We suggest that BChl synthesis in Osc. trichoides proceeds via the C-5 pathway, similar to representatives of other families of green bacteria (Chlorobium limicola and Chloroflexus aurantiacus). We demonstrated that the BChl c: BChl a ratio in the chlorosomes varied from 55 : 1 to 110 : 1, depending on light intensity. This ratio is, therefore, closer to that of Chlorobiaceae, and it significantly exceeds the BChl c: BChl a ratio in Chloroflexaceae.

Purification and Characterization of the B808–866 Light-harvesting Complex from Green Filamentous Bacterium Chloroflexus aurantiacus

The integral membrane light-harvesting complex B808-866 from the thermophilic green filamentous bacterium Chloroflexus aurantiacus has been isolated and characterized. Reversed-phase HPLC analysis demonstrated that the number of bacteriochlorophyll (BChl) in the B808-866 antenna complex is 36 +/- 2 per reaction center. The main carotenoid type is gamma-carotene, and the molar ratio of BChl to carotenoid is 3:2. The steady-state absorption and fluorescence spectroscopy of the B808-866 complex are reminiscent of the well-studied LH2 peripheral antenna of purple bacteria, whereas the protein sequence and the circular dichroism spectrum of B808-866 is more similar to the LH1 inner core antenna. The efficiency of excitation transfer from carotenoid to BChl is about 25%. The above results combined with electron microscopy and dynamic light scattering analysis suggest that the B808-866 antenna is more like the LH1, whereas surrounds the reaction center but probably consists of 24 building blocks with a ring diameter of about 20 nm. The above results suggested that there are probably two reaction centers inside the ring of B808-866. The unique properties of this light-harvesting complex may provide insights on the protein-pigment interactions in bacterial photosynthesis.

Excitation energy transfer in individual light-harvesting chlorosome from green photosynthetic bacterium Chloroflexus aurantiacus at cryogenic temperature

The excitation energy transfer from bacteriochlorophyll(BChl)- c self-aggregates to energy-accepting BChl- a in proteins (baseplates) in an individual photosynthetic light-harvesting complex (chlorosome) of a green filamentous photosynthetic bacterium Chloroflexus aurantiacus was successfully observed at cryogenic temperature. The ratio of intensity of the fluorescence peak of BChl- a to that of BChl- c self-aggregates in individual chlorosomes, which demonstrated relative efficiency of the excitation energy transfer, was heterogeneous between 0.09 and 0.72. This suggests that excitonic interaction between BChl- c self-aggregates and BChl- a in baseplates was heterogeneous among individual chlorosomes.

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.